Coronaviruses, a genus in the family of Coronaviridae, are large, enveloped plus strand RNA viruses. The genomic RNA is 27 to 32 kb in size, capped and polyadenylated. Three serologically distinct groups of coronaviruses have been identified. Within each group, viruses are identified by hosts range and genome sequence. Coronaviruses have been identified in mice, rats, chickens, turkeys, swine, dogs; cats, rabbits, horses, cattle and humans (39, 40). Most coronaviruses infect only one host species, and can cause severe disease including gastroenteritis, and respiratory tract diseases. In humans, 3 coronaviruses have been studied in detail. HCoV-229E and HCo\7-0C43 have been identified in the mid sixties and are known to cause common cold (13-17 19, 41, 42). Besides common cold it has been suggested that the HCoV may cause a more serious disease in infants as HCoV-229E virus has been isolated from infants suffering from lower respiratory tract disease (28). The third and most recently identified coronavirus: SARS-CoV, is, with its ability to cause a life threatening pneumonia (43), the most pathogenic human coronavirus identified thus far. It has been suggested that SARS-CoV is the first member of a fourth group of coronaviruses, or that the virus is an outlier of the group 2 coronaviruses (27, 44).
The coronavirus genome encodes four structural proteins: the spike protein, the membrane protein, the envelope protein and the nucleocapsid protein. Several non-structural proteins are involved in replication and transcription, which are encoded by two long overlapping open reading frames (ORFs) at the 5′end of the genome (1A and 1B). These 2 ORFs are connected via a ribosomal frame shift. The polypeptides encoded by ORF 1A and 1B are post-translationally processed by viral encoded proteases. Furthermore, additional non-structural proteins are encoded between the S and E gene, or between the M and N gene or downstream of the N gene. Some of these “accessory non-structural protein genes” have been found to be not essential for virus reproduction (45, 46). The coronavirus gene products of 1A and 1B are translated from the genomic RNA but the remaining viral proteins are translated from subgenomic mRNAs (sg mRNA), each with a 5′end derived from the 5′ part of the genome. The sg mRNA are derived via a discontinuous transcription process that most probably occurs during negative strand synthesis (47). Discontinuous transcription requires base-pairing between cis-acting elements, the transcription associated sequences (TRSs), one located at the 5′ part of the genome (the leader TRS) and others located upstream of the OCRFs (the body TRSs) (48)).
The novel coronavirus that we present here was isolated from a child suffering from bronchiolitis. Infection by this virus was not an isolated case since we found 7 more persons suffering from respiratory tract disease carrying the virus. In addition, we show here the complete genome sequence providing critical information concerning the genome structure of the new coronavirus.
To date there is a range of human diseases with unknown etiology. For many of these a viral origin has been suggested, emphasizing the importance of a continuous search for new viruses22, 23, 24. Major difficulties are encountered when searching for new viruses. First, some viruses do not replicate in vitro, at least not in the cells that are commonly used in viral diagnostics. Second, for those viruses that do replicate in vitro and that cause a cytopathic effect (CPE), the subsequent virus-identification methods may fail. Antibodies raised against known viruses may not recognize the cultured virus and virus specific PCR methods may not amplify the new viral genome. We have developed a method for virus discovery based on the cDNA amplified restriction fragment length polymorphism technique (cDNA-AFLP). With this technique, RNA or DNA is reproducibly amplified. There is no need to have prior knowledge of the sequence of the target gene1. Generally the cDNA-AFLP method is used to monitor differential gene expression, however, we modified this method such that it can amplify viral sequences either directly from patient blood-plasma/serum samples or indirectly from CPE-positive virus culture (FIG. 1). In the modified Virus-Discovery-cDNA-AFLP (VIDISCA) method the mRNA isolation step prior to amplification is replaced by a treatment to selectively enrich for viral nucleic acid. Of relevance to the purification is a centrifugation step to remove residual cells and mitochondria. In addition, a DNAse treatment can be used to remove interfering chromosomal and mitochondrial DNA from degraded cells whereas viral nucleic acid is protected within the viral particle. Finally, by choosing frequently cutting restriction enzymes, the method can be fine-tuned such that most viruses will be amplified.
In January 2003 a 7-month-old child appeared in the hospital with coryza, conjunctivitis and fever. Chest radiography showed typical features of bronchiolitis and a nasopharyngeal aspirate specimen was collected (sample nr: NL63) five days after the onset of disease. All diagnostic tests on this sample for respiratory syncytial virus (RSV), adenovirus, influenza A and B virus, parainfluenza virus type 1, 2 and 3, rhinovirus, enterovirus, HCoV-229E and HCoV-OC43 were negative. Immunofluorescent assays to detect RSV, adenovirus, influenza A and B virus, and parainfluenza virus type 1, 2 and 3 in cultures of the virus remained negative. Acid lability and chloroform sensitivity tests demonstrated that the virus was most likely enveloped and not a member of the Picornavirus group. In fact it was a new coronavirus.
In the present invention we present a detailed description of a novel human coronavirus. Coronaviruses are characterized by a very long non-segmented, single-stranded, (+) sense RNA of approximately 27-31 kb. This is the longest genome of any known RNA virus. The genome has a 5′ methylated cap and 3′ poly-A and functions directly as mRNA. Thus far only 3 human coronaviruses have been characterized, therefore sorting out the characteristics of a fourth human coronavirus supplies attractive information on the variation among the human coronaviruses. The novel virus is a member of the group 1 coronaviruses and is most related to HCoV-229B, yet the differences are prominent. The similarity is not larger than 85% at the nucleotide level, at the position of the 4A and 4B gene of HCoV-229E only one ORF is present in HCoV-NL63 (ORF 3), and the 5′ region of the S gene of HCoV-NL63 contains a unique in frame insertion of 537 nucleotides. Since binding of the receptor has been mapped to the N-terminal part of the protein, the 179 amino acids encoded by the insertion are most likely involved in receptor binding. This unique part at the N-terminus of the spike protein might explain the expanded host range of the virus in cell culture. Where HCoV-229E is fastidious in cell culture with a narrow host range, HCoV-NL63 replicates efficiently in monkey kidney cells. Besides HCoV-NL63 also SARS-CoV is able to replicate in monkey kidney cells (Vero-E6 cells and NCI-H292 cells for SARS-CoV (21)). Yet, comparing the predicted Spike genes did not identify a protein region that is shared by both viruses to clarify the common host range of the viruses in vitro. Also the insertion in the S gene of HCoV-NL63 was not present in the SARS S gene. Alternatively, other viral proteins may be involved in the cell tropism of a virus, however we did not identify any gene of HCoV-NL63 that had more similarity at the protein level to the SARS-CoV than to the similarity to HCoV-229E.
The 2 major differences between HCoV-229E and HCoV-NL63: the insertion in the S gene and the altered non-structural accessory proteins genes, are comparably to the differences that are noted between the porcine coronaviruses PRCoV and TGEV. Although these 2 porcine viruses are antigenically and genetically related their pathogenicity is very different. TGEV causes severe diarrhea with a high mortality in neonatal swine. It replicates and destroys the enterocytes in the small intestine whereas PRCoV has a selective tropism for respiratory tissue with very little to no replication in intestinal tissue. The genome differences in the S, 3A and 3B genes between TGEV and PRCoV are comparable with the differences between HCoV-NL63 and HCoV-229E. Alike HCoV-NL63, TGEV has a unique in frame insertion at the 5′ part of the S gene ranging from 672 to 681 nt (53). Furthermore, the accessory protein genes 3A and 3B that are intact in TGEV, are often mutated or inactive in the PRCoV. Extrapolating these data to the human coronaviruses one can speculate that HCoV-NL63 might be a more pathogenic human virus in comparison with HCoV-229E. However there are no epidemiological data supporting this. Based on our data it seems likely that HCoV-NL63 and HCoV-229E share the same pathogenicity. The common cold virus HCoV-229E can cause a more serious disease in infants (28), comparable to our data that suggest that HCoV-NL63 is causing a respiratory disease only in infants and immuno-compromised patients.
To date, a viral pathogen cannot be identified in a substantial portion of respiratory disease cases in humans (on average 20%59), our data indicate that in a part of these cases HCoV-NL63 is involved. The frequency with which HCoV-NL63 was detected in patients suffering from respiratory disease was up to 5% in January 2003. The virus was not detected in any of the samples collected in the spring or summer of 2003, which is in harmony with the epidemiology of human coronaviruses that have a tendency to spread predominantly in the winter season (15). The primers for our diagnostic PCR were located in the 1B gene and the genomic RNA can be used as template. Using primers that anneal in the nucleocapsid gene or 3′UTR supplies more template in the PCR because besides the genomic RNA also all sg mRNA in infected cells are template for amplification. It might be that the number of persons that we found positive for HCoV-NL63 is an underestimation of the correct number of persons carrying HCoV-NL63.
The newly found coronavirus, (designated HCoV-NL63) was characterized and sequenced. A sequence of a prototype HCoV-NL63 is provided in FIG. 19 and parts thereof in table 3. In one aspect the invention therefore provides an isolated and/or recombinant nucleic acid comprising a sequence as depicted in FIG. 19 and/or table 3, or a functional part, derivative and/or analogue thereof. The virus HCoV-NL63 is characterized by the prototype, however, many natural variants exist as for instance shown in FIG. 16 for polymorphisms in the ORF 1a region. The existence of such natural variants is normal for RNA viruses that undergo frequent mutation through for instance the introduction of mistakes by the polymerases that copy the genome. HCoV-NL63 viruses that have a slightly divergent nucleic acid sequence are thus also provided by the present invention. Such viruses are considered to be a derivative of the nucleic acid having the prototype nucleic acid sequence. The variant does not necessarily have to be a natural variant. It is very well possible to generate variants through recombinant means. For instance many parts of the virus can be altered through nucleotide substitution to make use of the redundancy in the triplet genetic code for particular amino acids. Thus without altering the amino acid sequence of the encoded proteins. However, even amino acid alterations can typically be introduced without affecting the replicating and coding potential of the viruses. For instance conservative amino acid substitutions are often tolerated. Alterations in the prototype virus may be up to 70% of the nucleic acid sequence without altering the replicating potential of the virus. Thus in one embodiment the invention provides an isolated and/or recombinant nucleic acid that is at least 70% homologous to a nucleic acid of the prototype RCoV-NL63. Most of the viable variants however are at least 95% homologous and more preferably at least 99% to a nucleic acid according to the prototype HCoV-NL63. The homology between different coronaviruses in the UTR regions is typically high, for this reason the homology in this application is measured in a region outside the UTR regions, preferably in a protein coding region. Thus the invention provides a derivative of HCoV-NL63 virus comprising at least 95% homology and preferably at least 99% homology (on the nucleic acid level) in at least one protein coding region depicted FIG. 20, 21, 22, 23, or table 3. The nucleic acid of the virus or parts thereof can be cloned and used as a probe to detect the virus in samples. Thus the present invention further provides an isolated and/or recombinant nucleic acid comprising a stretch of 100 consecutive nucleotides of a nucleic acid of the prototype virus, or a region that is at least 95% and preferably at least 99% homologous to said 100 consecutive nucleotides(when measured on the nucleic acid level outside a UTR region). A stretch of 100 consecutive nucleotides is considered to be a functional part of the virus of the present invention. Further provided is a bacterial vector comprising a nucleic acid of HCoV-NL63 or a functional part, derivative and/or analogue thereof. Further provided is a bacterium comprising said bacterial vector. The sequence of HCoV-NL63 or a part thereof can be used to generate a primer that is specific for HCoV-NL63 and thus capable of specifically replicating HCoV-NL63 nucleic acid. Similarly, a probe can be generated that specifically hybridizes to HCoV-NL63 nucleic acid under stringent conditions. Thus the invention further provides a primer and/or probe, capable of specifically hybridizing to a nucleic acid of a HCoV-NL63 virus or functional part, derivative or analogue thereof. Preferably, said primer or probe is capable of hybridizing to said nucleic acid under stringent conditions. In a particularly preferred embodiment said primer and/or probe comprises a sequence as depicted in table 3, table 7, table 10 or FIGS. 16 to 18. The nucleic acid of the prototype virus encodes various proteins and poly-proteins. These proteins are expressed for instance in cells producing the virus or transformed with a nucleic acid encoding the (poly)protein. The invention thus further provides an isolated and/or recombinant proteinaceous molecule comprising a sequence as depicted in FIG. 20, 21, 22, 23 or table 8, or a functional part, derivative and/or analogue thereof. Many different variants of the proteins having the same function in kind, not necessarily in amount are, as mentioned above, present in nature and can be generated artificially, thus the invention further provides an isolated and/or recombinant proteinaceous molecule that is at least 70% homologues to a proteinaceous molecule mentioned above. Such homologous proteins are considered derivatives of a protein encoded by the prototype. Preferably, a derivative protein comprises at least 95% and more preferably at least 99% homology with a protein encoded by the prototype HCoV-NL63. Fragments and parts of a proteinaceous molecule encoded by the prototype virus can be generated, such parts are therefore also provided by the present invention. In a preferred embodiment is provided an isolated and/or recombinant proteinaceous molecule comprising a stretch of at least 30 consecutive amino acids of a proteinaceous molecule encoded by the prototype virus. A protein encoded by the prototype virus can be encoded through a variety of different nucleic acid sequences using the redundancy of the genetic code. Thus the invention further provides a nucleic acid encoding a protein depicted in FIG. 20, 21, 22, 28 or table 3. The HCoV-NL63 virus can be replicated using in vitro growing cell lines. The virus can be harvested from such cultures and used in a variety of different application including but not limited to the generation of an immune response in a subject. The invention thus further provides an isolated or recombinant virus comprising a HCoV-NL63 nucleic acid sequence or a functional part, derivative and/or analogue thereof. Also provided is an isolated or recombinant virus comprising a proteinaceous molecule as depicted in FIG. 20, 21, 22, 23 or table 8, or a functional part, derivative and/or analogue thereof. Subjects that have become infected with HCoV-NL63 can display a number of different clinical and/or subclinical symptoms. Thus further provided is an isolated or recombinant virus or a functional part, derivative or analogue thereof capable of inducing a HCoV-N63-related disease. The virus comprises substances that can be used to generate specific binding partners that are able to specifically bind the substance of the virus. Binding partners can be generated by means of injection of the virus into in an immuno-competent subject. As a result of the immunization the serum obtained from the subject will typically contain a number of different antibodies specific for the virus or an immunogenic part, derivative and/or analogue thereof. Specific binding partners can of course be generated through a large variety of different technologies. For instance phage display technologies. The method of producing the specific binding partner is not limited herein. The binding is typically specific for a proteinaceous part of the virus. But can of course also be specific for a virus specific post translation modification of a protein contained in the virus. Thus the present invention further provides an isolated binding molecule capable of specifically binding a proteinaceous molecule of a HCoV-NL63 virus preferably against encoded by a nucleic acid of the prototype HCoV-NL63. Preferably, a proteinaceous molecule as depicted in FIG. 20, 21, 22, 23 or table 3, or a functional part, derivative and/or analogue thereof. The binding molecule can be capable of specifically binding a nucleic acid sequence of a HCoV-NL63, preferably of FIG. 19 or table 3. The binding molecule is preferably a proteinaceous molecule. However, other binding molecules are also within the scope of the present invention. For instance, it is possible to generate protein mimetics or analogues having the same binding quality as a protein in kind not necessarily in amount. Provided is further a method for producing a binding molecule according to the invention comprising                producing molecules capable of binding a HCoV-NL63 virus or functional part, derivative or analogue thereof or an isolated and/or recombinant proteinaceous molecule encoded by a prototype nucleic acid of HCoV-NL63, and        selecting a proteinaceous binding molecule that is specific for said virus and/or said proteinaceous molecule.        
The overall homology of HCoV-NL63 virus with other human coronaviruses is not very high. Thus many different binding molecules capable of specifically binding to HCoV-NL63 virus can be generated. Such binding molecules can be used to detect HCoV-NL63 virus in a sample. The invention thus further provides an isolated or recombinant virus which is immunoreactive with a binding molecule capable of specifically binding HCoV-NL63 virus. Similarly, the invention provides the use of an isolated and/or recombinant proteinaceous molecule as depicted in FIG. 20, 21, 22, 23 or table 3, or a functional part, derivative and/or analogue thereof, for detecting a binding molecule capable of specifically binding HCoV-NL63 virus, or functional part, derivative and/or analogue of said virus in a sample Vise versa, HCoV-NL63 virus can be used to detect a molecule capable of specifically binding said virus in a sample. Binding of HCoV-NL63 virus to a susceptible target cell occurs via a specific receptor. This receptor can be used as a binding molecule of the invention. Preferably, the binding molecule comprises an antibody or functional equivalent thereof. The detection methods can be used to diagnose HCoV-NL63 related disease in a subject. Thus provided is a method for detecting a HCoV-NL63 virus or functional part, derivative or analogue thereof in a sample, comprising hybridizing and/or amplifying a nucleic acid of said virus or functional part, derivative or analogue with a HCoV-NL63 specific primer and/or probe and detecting hybridized and/or amplified product. Further provided is a kit, preferably a diagnostic kit comprising a HCoV-NL63 virus or functional part, derivative or analogue thereof a binding molecule according to the invention, and/or a HCoV-NL63 virus specific primer/probe according to invention.
In a particular preferred embodiment is provided the use of a primer or probe capable of specifically hybridizing to a nucleic acid of a HCoV-NL63 virus or functional part, derivative or analogue thereof or a binding molecule capable of specifically binding a proteinaceous molecule depicted in FIG. 20, 21, 22, 23 or table 3 or an HCoV-NL63 virus and/or a nucleic acid or functional part, derivative or analogue of a prototype HCoV-NL63 for detecting and/or identifying a HCoV-NL63 coronavirus in a sample. Preferably said nucleic acid comprises a sequence as depicted in table 3.
The invention further provides a vaccine comprising HCoV-NL63 virus or functional part, derivative or analogue thereof. Further provided is a vaccine comprising a proteinaceous molecule depicted in FIG. 20, 21, 22, 23 or table 3 or functional part, derivative and/or analogue of such a proteinaceous molecule. A proteinaceous molecule of the invention may be provided as a vaccine by itself or as a part of the protein or as derivatives or analogues thereof. A suitable analogue is a nucleic acid encoding a HCoV-NL63 virus proteinaceous molecule or a functional part or derivative thereof. The nucleic acid may be used in a DNA vaccine approach which is also provided in the present invention. As carrier for the DNA vaccine it is often suitable to incorporate an expressible HCoV-NL63 virus nucleic acid in a viral replicon allowing replication of the HCoV-NL63 virus nucleic acid in the target cell and thereby allowing boosting of the provided immune response. A HCoV-NL63 virus encoded protein that is suited for such a DNA vaccine approach is the S protein depicted in FIG. 22 or a functional part, derivative and/or analogue thereof. A part of an S protein preferably comprises an immunogenic part of the 537 in frame insertion as compared with HCoV-229E virus. Preferably said part comprises essentially said 537 insertion. With the 537 insertion is meant a sequence corresponding to sequences 20472 to 21009 of FIG. 19. Other suitable candidates are the M and or the N protein or a functional part, derivative and/or analogue thereof. Typically a vaccine includes an appropriate adjuvant. Apart from the use in a vaccine the mentioned virus and/or proteinaceous molecules can also be used to generate and/or boost a HCoV-NL63 virus specific immune response in a subject. The immune response can be both cellular or humoral. Thus further provided is an isolated T-cell comprising a T-cell receptor that is specific for HCoV-NL63 virus or a proteinaceous molecule encoded by a prototype HCoV-NL63 virus. Further provided is an isolated B-cell producing an antibody specific for HCoV-NL63 virus or a proteinaceous molecule encoded by a HCoV-NL63 virus. The antibody or T-cell receptor can be cloned whereupon a cell line can be provided with an expression cassette comprising the cloned receptor or antibody. Thus the invention further provides a cell producing such a receptor or antibody. Such a cell is preferably a cell that is suitable for large scale production of the mentioned proteins such as CHO cells.
It is also possible to provide a subject with passive immunity to HCoV-NL63 virus. To this end the subject can be provided with a HCoV-NL63 specific binding molecule of the invention. Such immunity can be used to provide a barrier for (further) infection with HCoV-NL63 virus in the subject, thus further provided is a vaccine comprising a HCoV-NL63 virus specific binding molecule according to the invention. In a preferred embodiment, passive immunity is provided by a human or humanized antibody capable of specifically binding a HCoV-NL63 virus of the invention. The barrier does not have to be perfect. The presence of a binding molecule at least reduces the spread of the virus to other target cells in the subject. The passive immunity may be administered to a subject as prophylactic to at least reduce the spread of HCoV-NL63 virus in the subject when exposed to the virus. Alternatively, the passive immunity may be provided to a subject already infected with the virus. In the latter case one or more HCoV-NL63 virus specific binding molecules of the invention are used as a medicament to at least reduce the spread of the virus in the subject and thereby at least in part combat the virus infection. The invention thus further provides a medicament comprising a HCoV-NL63 virus specific binding molecule according to the invention. Further provided is the use of a virus of the invention or functional part, derivative or analogue thereof or a proteinaceous molecule of the invention or a HCoV-NL63 virus specific binding molecule of the invention, for the preparation of a vaccine against a coronaviral genus related disease. Further provided is a method for treating an individual suffering from, or at risk of suffering from, an HCoV-NL63 related disease, comprising administering to said individual a vaccine or medicament according to the invention. In yet another embodiment is provided a method for determining whether an individual suffers from an HCoV-NL63 related disease, comprising obtaining a sample from said individual and detecting a HCoV-NL63 virus or functional part, derivative or analogue thereof in said sample.
In yet another embodiment is provided an isolated cell, or recombinant or cell line comprising HCoV-NL63 virus, or a functional part, derivative and/or analogue thereof. Preferably said cell is a primate cell, preferably a monkey cell. In a preferred embodiment, said cell is a cell that replicates the HCoV-NL63 virus of the invention. In a particular embodiment the cell is a kidney cell. The cell can be used to produce the HCoV-NL63 virus of the invention or to attenuate HCoV-NL63 such that it becomes less pathogenic. Virus attenuation is spontaneous upon continued culture of the virus on the mentioned preferred cell lines. Attenuated HCoV-NL63 virus can be used as a vaccine.
HCoV-NL63 virus encodes an endoprotease. A sequence for the protease in the prototype
HCoV-NL63 virus is depicted in FIG. (21). The protease is important for the processing of the polyproteins encoded by HCoV-NL63. The action of the protease is at least in part inhibited by a viral protease inhibitor as further described herein. Thus the invention further provides a compound for at least in part inhibiting HCoV-NL63 virus replication. Preferred compounds are inhibitors of inosine monophosphate dehydrogenase (55) (e.g. Ribavirin(54) and mycophenolic acid), orotidine-5′-phosphate decarboxylase inhibitors (e.g. 6-azauridine and pyrazofurin), 3CL-protease inhibitors(56) (e.g. the VNSTLQ (SEQ. ID. NO: 1)-AG7088 ester, see below), cap-methylase inhibitors(58) (carboxylic adenosine analogs e.g. Neoplanocin A and 3-deazaneoplancin A), nitrous oxide synthase inducing compounds (e.g. glycyrrhizin) and Interferons (57). Of these the protease inhibitors are particularly preferred. The sequence VNSTLQ (SEQ. ID. NO: 1) is the N-terminal proteolytic processing site of SARS-3CLpro that is used in the 3Clpro inhibitor VNSTLQ (SEQ. ID. NO: 1)-AG7088 (56). In this compound the hexapeptide VNSTLQ (SEQ. ID. NO: 1) is C-terminally linked to the vinylogous ethyl ester (AG7088, see structural formula 1 depicted below,) that inhibits SARS 3CLpro activity.

The hexapeptide VNSTLQ (SEQ. ID. NO: 1) corresponds to YNSTLQ (SEQ. ID. NO: 2)in HCoV-NL63. Therefore YNSTLQ (SEQ. ID. NO: 2)- AG7088 inhibits the HCoV-NL63 3CLpro orthologs. Thus in a preferred embodiment the protease inhibitor comprises the amino acid sequence VNSTLQ (SEQ. ID. NO: 1) more preferably YNSTLQ (SEQ. ID. NO: 2). Analogues of such protease inhibitors that comprise the same activity in kind not necessarily in amount are also provided by the present invention. Such analogues include, compounds comprising a peptide with the preferred sequence, wherein the peptide comprises a modification. Other analogues include compounds having protein mimetic activity that mimic the preferred amino-acid sequence. S-adenosylmethionine-dependant ribose 2′-orthomethyltransferase Plays a role in the methylation of cap structure (GpppNm) at the 5′end of the viral RNA. Antiviral compounds inhibiting this transfer of methyl groups to reaction (carboxylic adenosine analogs e.g. Neoplanocin A and 3-deazaneoplancin A) interfere with expression of viral proteins.
The invention further provides a proteinaceous molecule encoded by HCoV-NL63 nucleic acid, wherein said proteinaceous molecule is a 3CL protease or a functional equivalent thereof. Functional equivalents include an proteolytically active part and/or derivative having one or more conservative amino acid substitutions. There are many methods known in the art to determine whether a compound has anticoronaviral activity, preferably antiproteolytic activity of a coronavirus. The invention thus further provides a method for determining whether a compound comprises anticoronavirus replication activity characterized in that said method utilizes HCoV-NL63-virus or a HCoV-NL63 protein involved in replication of HCoV-NL63 or a functional part, derivative and/or analogue thereof. Preferably, the invention provides a method for determining whether a compound is capable of at least in part inhibiting a viral protease characterized in that said protease is a 3CL protease of HCoV-NL63 or a functional part, derivative and/or analogue thereof. Preferred compounds that can be tested for 3CL inhibiting quality are hexapeptides located N-terminally of 3Clpro cleavage sites. Compounds effective in at least in part inhibiting 3Cl proteolytic activity can be used for the preparation of a medicament for the treatment of an individual suffering or at risk of suffering from a HCoV-NL63 virus infection.
One or more of the preferred anticoronaviral replication compounds can be used as a medicament for the treatment of a subject suffering from or at risk of suffering from a HCoV-NL63 virus infection. The invention thus further provides a medicament for the treatment of an individual suffering from an coronavirus infection or an individual at risk of suffering there from comprising wherein said coronavirus comprises a nucleic acid sequence of a HCoV-NL63 prototype virus or a functional part, derivative and/or analogue thereof.
In the present invention several different recombinant viruses are produced using HCoV-NL63 virus nucleic acid as a backbone. Such replication competent or replication defective recombinant virus can be used for instance as gene delivery vehicles. On the other hand parts of a HCoV-NL63 virus can be used in gene delivery vehicles that are based on other means for delivering genetic material to a cell. Thus the invention further provides a gene delivery vehicle comprising at least part of a HCoV-NL63 virus nucleic acid. Preferably of the prototype virus. Preferably comprising a nucleic acid encoding a protein of HCoV-NL63 virus or a functional part, derivative and/or analogue thereof. The invention also shows chimearic coronaviruses comprising nucleic acid derived from at least two coronaviruses wherein at least one of said parts is derived from a HCoV-NL63 virus. Said HCoV-NL63 virus derived part comprises preferably at least 50 nucleotides of a protein coding domain. More preferably said HCoV-NL63 derived part comprises at least 500 and more preferably at least 1000 nucleotides of the sequence as depicted in FIG. 19 or a functional derivative thereof. In a preferred embodiment the invention provides a chimearic coronavirus comprising at least 1000 nucleotides of a sequence as depicted in FIG. 19 and at least 1000 nucleotides of another coronavirus wherein said latter 1000 nucleotides comprise a sequence that is more than 5% sequence divergent with a sequence as depicted in FIG. 19. The sequences of a number of HCoV-NL63 virus fragments are depicted in table 3. The location of the fragments in the large genomic RNA is depicted in FIG. 5. The invention therefore, in one aspect, provides an isolated or recombinant virus comprising a nucleic acid sequence as depicted in table 3, or a functional part, derivative or analogue of said virus. With the aid of the identifying prototype fragments it is possible to further sequence the genome. One way of doing this by primer walking on the genome. A primer is directed to a region of which the sequence is known and this primer is used to sequence a flaking region that is as yet unknown. A subsequent primer can be generated against the newly identified sequence and a further region can be sequenced. This procedure can be repeated until the entire sequence of the virus is elucidated. As a source of the virus one may turn to Dr. C. van der Hoek, Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Alignments of the determined nucleic acid sequences revealed the reading frame used in the sequences found, accordingly the invention further provides an isolated or recombinant virus comprising an amino acid sequence as depicted in (table 3). or a functional part, derivative or analogue of said virus. A particular amino acid sequence can be produced from a variety of nucleic acids depending on the codons used. Thus the invention further provides a nucleic acid encoding an amino acid sequence as depicted in (table 3). Further provided is an isolated or recombinant virus comprising a nucleic acid sequence encoding an amino acid sequence as depicted in (table 3 ), or a functional part, derivative or analogue of said virus.
Coronaviruses as many other types of viruses acquire a plurality of spontaneous and selected mutations upon spreading of the virus through the subject population and/or during culturing ex vivo. Moreover, artificial mutations having no recognized counterpart in nature can be introduced into the sequence of the prototype virus or a derivative thereof, without altering the viral- and/or disease causing properties of the virus. Having characterized the prototype of the newly discovered subtype gives access to this group of viruses belonging to the same subtype. Thus the invention further provides an isolated or recombinant virus comprising a nucleic acid sequence that is approximately 80% homologous to a sequence as depicted in table 3, or 80% homologous to an amino acid sequence depicted in Table 3 (. Preferably the homology is at least 90%, more preferably at least 95% and even more preferably at least 99%.
The respective prototype fragments were compared with a database of viral sequences and hits having a particularly high homology are mentioned in the tables 5 and 6. It may be noted that the compared fragments do not share extensive homology with any of the currently known Coronaviruses. The invention thus provides an isolated and/or recombinant virus comprising an amino acid sequence which is more than 89% homologous to 163-2 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 60% homologous to 163-4 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising a nucleic acid sequence which is more than 85% homologous to 163-9 nucleic acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 94% homologous to 163-10 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 50% homologous to 163-11 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%.
Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 87% homologous to 163-14 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 88% homologous to 163-15 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising an amino acid sequence which is more than 78% homologous to 163-18 amino acid sequence as depicted in Table 3. Preferably said homology is at least 90%, more preferably at least 95% and even more preferably at least 99%. Further provided is an isolated or recombinant virus comprising a nucleic acid sequence which is at least 50% homologous to a nucleic acid sequence as depicted in Table 3. Preferably said homology is at least 80%, more preferably at least 90%, more preferably at least 95% and even more preferably at least 99%.
The invention also provides a functional part, derivative and/or analogue of an isolated and/or recombinant HCoV-NL63 virus. A part of a virus can be a membrane containing part, a nucleocapsid containing part, a proteinaceous fragment and/or a nucleic acid containing part. The functionality of the part varies with the application chosen for the part, for instance, part of the virus may be used for immunization purposes. In this embodiment the functionality comprises similar immunogenic properties in kind as the entire virus not necessarily in amount. Another use of the virus is the infectivity of the virus, for instance, for in vitro (or in vivo) culture, in this embodiment the functionality comprises a similar infectivity in kind not necessarily in amount. Many other functionalities may be defined, as there are many different uses for viruses, non-limiting examples are the generation of chimeric viruses, (i.e. with one or more other (corona) viruses, and the generation of viral vectors for vaccination and/or gene therapeutic purposes. Such viruses and/or vectors also contain a functional part of HCoV-NL63 and are thus also encompassed in the present invention. A functional derivative of a virus of the invention is defined as a virus that has been altered such that the properties of said compound are essentially the same in kind, not necessarily in amount. A derivative can be provided in many ways, for instance through nucleotide substitution (preferably “wobble” based), through (conservative) amino acid substitution, subsequent modification, etcetera.
Analogous compounds of a virus can also be generated using methods in the art. For instance, a chimeric virus can be produced, or an HCoV-NL63 virus having a chimeric protein. For instance, HCoV-NL63 can be rendered more immunogenic by generating a cell surface associated fusion protein comprising at least part of an HCoV-NL63 surface protein and a non-HCoV-NL63 immunogenic part. HCoV-NL63 virus comprising such chimeric protein can be used for inducing an enhanced immune response in a host, for instance for vaccination purposes.
As used herein, the term “a virus of the invention” is meant to also comprise a functional part, derivative and/or analogue of said virus.
The three groups of coronaviruses are associated with a variety of diseases of humans and domestic animals, including gastroenteritis and upper and lower respiratory tract disease. The human coronaviruses HCoV-229E and HCoV-OC43 are associated with mild disease (the common cold) but more severe disease is observed in children16, albeit at a very low incidence. Several coronaviruses cause a severe disease in animals and SARS-CoV is the first example of a coronavirus that causes severe disease in humans. However, it should be emphasized that a substantial part of respiratory disease cases in humans remains undiagnosed. For instance, a recent survey of respiratory viruses in hospitalized children with bronchiolitis in Canada could not reveal a viral pathogen in about 20% of the patients17. The fact that we identified the new coronavirus in a child with bronchiolitis shows that HCoV-NL63 is a pathogenic respiratory virus. When considering that the HCoV-NL63 is a pathogenic respiratory virus able to cause bronchiolitis in infected children, the interesting question remains why HCoV-NL63 was not recognized previously by cell culture. We found that the virus can be cultured in monkey kidney cells (tMK or LLC-MK2 cells), cells that are often used in a routine diagnostic setting and one might therefore speculate that HCoV-NL63, like SARS-CoV, was newly introduced from an animal reservoir into the human population or that this is a human virus that recently broadened its host cell range. Clearly it is of importance to study the prevalence of HCoV-NL63 infection, and screening specimens from patients with respiratory tract disease using the HCoV-NL63 diagnostic RT-PCR will shed light on this issue. It is remarkable that the new human coronavirus was harvested from tMK cells and LLC-MK2 cells since coronaviruses are typically fastidious in cell culture with a narrow host range. However, both SARS-CoV and HCoV-NL63 seem to replicate efficiently in monkey kidney cells (Vero-E6 cells and NCI-H292 cells for SARS-CoV). The recently described genome of SARS-CoV has several exclusive features, including some unique open reading frames that are probably of biological significance15, 18. We will therefore analyze the complete genome sequence of HCoV-NL63 to screen for similarities and differences with SARS-CoV that may determine the expanded host cell range and enhanced pathogenicity of these viruses.
HCoV-NL63 is associated with a particular phenotype in infected subjects. The phenotype can encompass bronchiolitis, coryza, conjunctivitis and fever and may further encompass other respiratory problems and diarrhea. In one embodiment the invention thus further provides an isolated and or recombinant virus of the invention (having one or more of the above mentioned homology) wherein said virus or functional part, derivative and/or analogue further comprises the capability to induce an HCoV-NL63 related disease or symptom in a subject. In another embodiment the invention provides an isolated and/or recombinant virus of the invention further comprising the property to cause CPE in tertiary monkey kidney cells (tMK; Cynomolgus monkey37) and/or upon passage onto the monkey cell line LLC-MK2 (ECCAC 85062804, ATCC CCL-7). In a preferred embodiment said virus does not produce CPE in Vero-cells (ATCC CRL-1586)34.
The invention further provides a nucleic acid as depicted in table 3, and an amino acid sequence as depicted in Table 3, or a functional part and/or equivalent of such a nucleic acid and/or amino acid sequence. A functional equivalent of said nucleic acid comprises the same hybridization properties in kind, not necessarily in amount, as said nucleic acid (or part thereof). A functional equivalent of an amino acid sequence of the invention comprises the same immunogenic properties in kind, not necessarily in amount, as said amino acid sequence (or part thereof). A part of a nucleic acid of the invention comprises at least 15 nucleotides, preferably at least 20, more preferably at least 30 nucleotides. A part of an amino acid sequence comprises at least 5 amino acids in peptidic linkage with each other, more preferably at least 8, and more preferably at least 12, more preferably at least 16 amino acids. In a preferred embodiment said nucleotides and/or amino acids are at least semi-consecutive, more preferably, said nucleotides and/or amino acids are consecutive. An equivalent of a nucleic acid and/or amino acid sequence of the invention or part thereof comprises at least 80% homology to a nucleic acid and/or amino acid sequence of the invention, preferably at least 90% homology, more preferably at least 95% and even more preferably at least 99% homology to a nucleic acid and/or amino acid sequence of the invention or a part thereof.
The invention further provides a primer and/or probe, capable of specifically hybridizing to a nucleic acid of a virus or functional part, derivative or analogue according to the invention, preferably a primer and/or probe, capable of specifically hybridizing to a nucleic acid sequence as depicted in Table 3. More preferably, a primer and/or probe, which is capable of hybridizing to said nucleic acid under stringent conditions. In a particular preferred embodiment is provided a primer and/or probe, comprising a sequence as depicted in Table 7.
The art knows many ways in which a specific binding member can be generated against an identified nucleic acid, lipid and/or amino acid sequence. Such specific binding members may be of any nature but are typically of a nucleic acid and/or proteinaceous nature. The invention thus further provides an isolated molecule capable of specifically binding a virus, nucleic acid and/or amino acid or functional part, derivative or analogue thereof according to the invention. Said isolated molecule is also referred to as specific binding member. Preferably said specific binding member is capable of specifically binding at least part of a nucleic acid sequence as depicted in table 8 and/or at least part of an amino acid sequence as depicted in Table 3. In a preferred embodiment said binding member is a proteinaceous molecule. Preferably an antibody or a functional part, derivative and/or analogue thereof. A specific binding member preferably comprises a significantly better binding property for the HCoV-NL63 virus compared to unrelated control. However, for instance for antibodies, it is possible that the epitope specifically recognized in HCoV-NL63 is also present in a limited number of other molecules. Thus though the binding of the binding member may be specific, it may recognize also other molecules than those present in HCoV-NL63. This cross-reactivity is to be separated from a-specific binding and is a general property of antibodies. Cross-reactivity does not usually hinder the selection of suitable specific binding members for particular purposes. For instance a specific binding member that also recognized a protein in liver cells can be used in many applications even in the presence of liver cells, where additional information such as location in the cell can often be used to discriminate.
One source of an antibody of the invention is the blood of the infected subjects screened for the virus of the present invention. One may further characterize B-cells obtained from said subject, A suitable B-cell may be cultured and the antibody collected. Alternatively, the antibody may be sequenced from this B-cell and generated artificially. Another source of an antibody of the invention can be generated by immunisation of test animals or using artificial libraries to screen a purified fraction of virus. A functional part of an antibody has essentially the same properties of said antibody in kind, not necessarily in amount. Said functional part is preferably capable of specifically binding an antigen of HCoV-NL63. However, said functional part may bind such antigen to a different extend as compared to said whole antibody. A functional part or derivative of an antibody for instance comprises a FAB fragment or a single chain antibody. An analogue of an antibody for instance comprises a chimeric antibody. As used herein, the term “antibody” is also meant to comprise a functional part, derivative and/or analogue of said antibody.
Once antibody of the invention is obtained, a desired property, such as its binding capacity, can be improved. This can for instance be done by an Ala-scan and/or replacement net mapping method. With these methods, many different proteinaceous molecules are generated, based on an original amino acid sequence but each molecule containing a substitution of at least one amino acid residue. Said amino acid residue may either be replaced by Alanine (Ala-scan) or by any other amino acid residue (replacement net mapping). Each variant is subsequently screened for said desired property. Generated data are used to design an improved proteinaceous molecule.
There are many different ways in which a specific binding member can be generated. In a preferred embodiment the invention provides a method for producing a specific proteinaceous binding member comprising producing proteinaceous molecules capable of binding a virus according to the invention or to a functional part, derivative or analogue, and selecting a proteinaceous molecule that is specific for said virus. If need be, the method may be used to generate a collection of proteinaceous molecules capable of binding to said virus or functional part, derivative and/or analogue thereof and selecting from said collection one or more binding members capable of specifically binding said virus or functional part, derivative and/or analogue thereof.
Any specific binding member is characteristic for the HCoV-NL63 virus of the invention. Thus a virus that is specifically reactive with such binding member is an HCoV-NL63 virus and thus provided by the invention. Thus the invention provides an isolated and/or recombinant virus that is immunoreactive with specific binding member of the invention, preferably a proteinaceous binding member. The invention further provides a composition of matter comprising isolated HCoV-NL63 virus, and/or a virus essentially corresponding to HCoV-NL63. The term, a virus “essentially corresponding to HCoV-NL63” refers to HCoV-NL63 viruses which are either identical to the HCoV-NL63 strain described hereinabove, or which comprises one or more mutations compared to the said HCoV-NL63strain. These mutations may include natural mutations or artificial mutations. Said mutations of course should allow detection with a specific binding member of HCoV-NL63, not necessarily with all of the specific binding members). Said mutations should allow the detection of the variants using common detection methods such as antibody interaction, amplification and/or hybridization.
Considering that specific binding members are important molecules for instance for diagnostic purposes, the invention further provides the use of a virus of the invention or functional part, derivative and/or analogue thereof, for detecting a molecule capable of specifically binding said virus in a sample. Further provided is the use of a nucleic acid and/or amino acid sequence of a virus or functional part, derivative or analogue as defined by the invention, or detecting a molecule capable of specifically binding said virus or functional part, derivative and/or analogue in a sample. Preferably said nucleic acid and/or amino acid sequence comprises a sequence as depicted in table 3 or Table 3 or a functional part, derivative or analogue thereof. Preferably said part is at least 30 nucleotides and/or amino acids long wherein said part preferably comprises more than 95% sequence identity, preferably more than 99%. In a preferred aspect said specific binding member comprises a specific ligand and/or antibody of said virus.
Further provided is a primer and/or probe according to the invention, a specific binding member of the invention, and/or a nucleic acid of a virus or functional part, derivative or analogue according to the invention, for detecting and/or identifying a HCoV-NL63 coronavirus or part thereof in a sample. Preferably, said nucleic acid comprises a sequence as depicted in table 3.
HCoV-NL63 virus may be used to generate an immune response in a subject. This can be useful for instance in vaccination strategies. Thus the invention further HCoV-NL63 provides HCoV-NL63 virus or functional part, derivative or analogue thereof for use as a vaccine or medicament. The medicament use is typically when the subject is already infected with the virus and the immunogen is used to augment the immune response against the virus. The invention further provides a specific binding member of the invention for use as a vaccine or medicament This use is particularly favorable for when the specific binding member comprises a proteinaceous molecule, preferably an antibody or functional part, derivative and/or analogue thereof. Such an antibody can provide passive immunity but may also have active components such as proteases attached to it. The medicament use may again be the case wherein a subject infected with an HCoV-NL63 virus is treated with the specific binding member.
Vaccines may be generated in a variety of ways. One way is to culture the HCoV-NL63 virus for example on the mentioned monkey cell line(s) and to use inactivated virus harvested from the culture. Alternatively, attenuated virus may be used either inactivated or as a live vaccine. Methods for the generation of coronavirus vaccines may be adapted to produce vaccines for the HCoV-NL63 of the invention. The invention thus further provides the use of an HCoV-NL63 virus or functional part, derivative or analogue thereof for the preparation of a vaccine against a coronaviral genus related disease. The invention further provides the use of a specific binding member of the invention for the preparation of a vaccine or medicament against a coronaviral genus related disease. Further provided is the use of an HCoV-NL63 virus or functional part, derivative or analogue thereof, a specific binding member of the invention, a nucleic acid of the invention or a primer and/or probe of the invention for diagnosis of a coronaviral genus related disease. Preferably said coronaviral genus related disease comprises a HCoV-NL63 coronavirus related disease.
Further provided is a vaccine comprising an HCoV-NL63 virus or functional part, derivative or analogue thereof and/or a specific binding member of the invention. Also provided is a medicament comprising an HCoV-NL63virus or functional part, derivative or analogue thereof and/or a specific binding member of the invention. Preferably said vaccine or medicament is used for at least in part preventing and/or treating a HCoV-NL63 related disease.
An important use of the present invention is the generation of a diagnostic tool for determining whether a subject is suffering from an HCoV-NL63 virus infection or has been exposed to an HCoV-NL63 virus infection. Many diff rent diagnostic applications can be envisioned. They typically contain an identifying component allowing the typing of the virus that is or was present in the subject. One diagnostic tool for HCoV-NL63 makes use of the particular proliferation characteristics of the virus in various cell lines. It replicates in the mentioned preferred monkey cell lines but does not replicate in Vero-cells. This property can be used to discriminate HCoV-NL63 from other known coronaviruses. Thus in one aspect the invention provides a diagnostic kit comprising at least one of the preferred monkey cell lines, preferably the tertiary monkey kidney cells (tMK; Cynomolgus monkey or the monkey cell line LLC-MK2.
Many modern diagnostic kits comprise a specific binding member (to detect the virus or virus infected cells) and/or an HCoV-NL63 virus or a functional part, derivative and/or analogue thereof and/or ammo acid of the invention or a functional part, derivative and/or analogue thereof (for detecting antibodies in blood components of the diagnosed subject). Many other current diagnostic kits rely on identification of HCoV-NL63 virus specific nucleic acid in a sample. There are various ways in which such an assay may be implemented one is a method for detecting an HCoV-NL63 virus or functional part, derivative or analogue thereof in a sample, comprising hybridizing and/or amplifying a nucleic acid of said virus or functional part, derivative or analogue with a primer and/or probe according to the invention and detecting hybridized and/or amplified product. The invention thus also provides a diagnostic kit comprising an HCoV-NL63 virus or functional part, derivative or analogue thereof, a specific binding member according to the invention and/or a primer/probe according to the invention.
Further provided is a method for treating an individual suffering from, or at risk of suffering from, a HCoV-NL63 related disease, comprising administering to said individual a vaccine or medicament according to the invention. Also provided is a method for determining whether an individual suffers from a HCoV-NL63 related disease, comprising obtaining a sample from said individual and detecting a HCoV-NL63 virus or functional part, derivative or analogue thereof in said sample with a method and/or diagnostic kit of the invention.
Further provided is an isolated or recombinant nucleic acid encoding a virus or functional part, derivative and/or analogue according to the invention and a nucleic acid according to the invention, comprising at least a functional part of a sequence as depicted in Table 3. Further provided is an amino acid sequence encoded by a nucleic acid according to the invention, and an amino acid sequence according to the invention, comprising at least a functional part of a sequence as depicted in Table 3. Further provided is a proteinaceous molecule capable of specifically binding HCoV-NL63, obtainable by a method according to the invention and, the use of such a proteinaceous molecule in a vaccine or a diagnostic method for the detection of HCoV-NL63.